To generate lentiviral vectors, we used calcium phosphate transfection of human embryonic kidney (HEK293) cells, as described by Tiscornia et al.26 Brief y, lentivirus packaging vectors RRE, REV2, VsVG and DNA of interest were added to a glass vial followed by 60 μl of 2.5 M calcium chloride. The solution was then totaled to 500 μl using deionized water. This mixture was then added dropwise to 500 μl 2× HBS solution while vortexing. The final solution was incubated at room temperature for 30 minutes and then added to a HEK293 culture plate at Day 0. DMEM from HEK293 culture on Day 1 was discarded. Media from Days 2 and 3 were collected and ultracentrifuged at 106,490 rcf (25,000 rpm) for 2 hours at 4°C Concentrated virus was then resuspended in 100 μl MEM and aliquoted for later use. All viruses were generated for expression under the TetOn promotor (Tet-On).
